Project abstract/summary This proposal responds to the NIH PA-16-157 ?New Technologies for the Glycosciences (R43/R44)?. We aim to develop efficient and streamlined cheomoenzymatic systems for glycan remodeling of antibodies and other important glycoproteins, through optimizing enzyme immobilization and simplifying the reaction procedures. Such immobilized enzymes, when combined with activated glycan library as kits, can help general academic and industrial users, particularly non-specialists, to prepare glycan-defined glycoproteins for structural and functional studies. The streamlined approach can also be applied in scale-up preparation of homogenous glycoproteins with therapeutic potential. Protein glycosylation is one of the most ubiquitous posttranslational modifications. It profoundly affects a protein?s properties such as folding, in vivo stability, immunogenicity, and pharmacokinetics, and also directly participate in a number of important biological processes, including cell adhesion, cancer progression, host-pathogen interactions, and immune responses. A major issue in glycoprotein studies is the structural heterogeneity and the difficulty in isolating homogeneous glycoforms for detailed structural and functional studies. Although significant progresses have been made in multiple approaches to prepare glycan-defined glycoprotein (such as total chemical synthesis and glycosylation through glycosylation pathway engineering in different host expression system), the pure glycoforms that can be achieved are still quite limited. To address this challenge, a novel chemoenzymatic method to produce homogeneous glycoprotein and glycopeptides has been developed recently. This convergent approach consists of two key steps: deglcosylation of glycoproteins with an endoglycosidase and subsequent attachment of a desired activated N-glycan to the protein-GlcNAc acceptor by a novel glycosynthase. In the proposed study, GlycoT Therapeutics attempts to streamline and simplify this enzymatic glycan remodeling method through immobilizing the enzymes and developing easy-to-use kits and protocols, which are particularly useful for non- specialists. Immobilization will offer multiple valuable benefits: 1) stabilizing the enzyme for sustained activity, repeated use, and easy storage; 2) removing the protein purification steps for protein-GlcNAc acceptor and final products, thus greatly simplifying the process; 3) eliminating the potential problem of wild type enzyme contamination from the deglycosylation step. To achieve these objectives, we propose to pursue the following two specific aims in Phase I study. Aim 1 is to immobilize key enzymes for glycan remodeling of antibodies, which include: the endoglycosidase S2 (Endo-S2) from Streptococcus pyogenes, the Endo-S2 glycosynthase mutant (D184M), and an ?1,6-fucosidase for defucosylation. Different methods of immobilization will be investigated. Aim 2 is to develop reaction kits and simply protocols for streamlined application of the enzymatic glycan remodeling of antibodies. In prospective phase II research, the study will expand to other endoglycosidases, to cover other glycoproteins/glycopeptides, for both academic and industrial applications.